Screen for gastric adenocarcinoma

ABSTRACT

It has been determined that a specific metaplastie lineage that contains immunoreactivity for a trefoil polypeptide, spasmolytic peptide, is associated with and gives rise to the vast majority of human adenocarcinomas. The identification of this Spasmolytic Polypeptide Expressing Metaplasia (SPEM) is a major factor for grading of biopsies of the stomach to assess risk for gastric cancer. It also forms the basis of a method for serological screening for those at risk for gastric cancer. In a preferred embodiment, antibodies to spasmolytic peptide (hSP) are used in immunostaining of biopsies of gastric tissue obtained by endoscopy for grading biopsies Those patients having these cells, characterized by a morphology more typical of a type of cell present normally in the intestine and not stomach, Brunner&#39;s gland cells, are at risk of developing adenocarinoma. Since these cells express hSP, antibodies or nucleic acid probes hybridizing to mRNA encoding hSP, can be used for rapid detection of the cells in tissue biopsies. The antibodies can also be used in serological tests for screening and following patients at risk for gastric cancer. In combination with evidence of previous or present infection with  H. pylori , the tests are predictive of the likelihood of developing adenocarcinoma.

BACKGROUND OF THE INVENTION

The United States government has certain rights in this invention byvirtue of a grant from the National Institutes of Health NIDDKD to JamesR. Goldenring.

Gastric cancer worldwide is the second highest cause of death fromcancer. While rates of gastric cancer have been decreasing in the UnitedStates, gastric cancer prevalence remains high in other parts of theworld, especially in Asia. Presently, patients in endemic regions forgastric cancer, especially Japan and China, undergo screening by upperendoscopy. There is no alternative at present for discovery of earlycancer other than by endoscopy. Grading of these endoscopies is basedsolely on hematoxylin and eosin staining. There are no prognosticmarkers that could indicate those at risk for future cancer development.Therefore, while biopsies can reveal early gastric cancers, patients inhigh risk regions who are initially negative by endoscopy must befollowed with endoscopies to rule out future developments.

The epidemiological association between chronic H. pylori infection andgastric carcinoma is now well established, such that the Working GroupMeeting of the International Agency for Research on Cancer with theWorld Health Organization recently classified Helicobacter pylori as agroup 1 human gastric carcinogen (Peura, D. A. Gastroenterology 113,S4-S8 (1997)). Parsonnet and colleagues reported that 84% of patientswith gastric cancer had serum antibodies against H. pylori, as opposedto 66% of uninfected matched controls (Parsonnet, et al. New Eng. J.Med. 325, 1127-1131(1991)). H. pylori infection induces a chronicgastritis progressing to atrophic gastritis, foveolar hyperplasia andintestinal metaplasia (Mobley, H. T. L. Gastroenterology 113, S21-S28(1997)). Recent studies have indicated that chronic gastritis associatedwith Helicobacter pylori contributes to the development of gastricadenocarcinoma (Peura, D. A. Gastroenterology 113, S4-S8 (1997);Parsonnet, et al. New Eng. J. Med. 325, 1127-1131 (1991); Forman et al.Br. J. Med. 302, 1302-1305 (1991); Normura, et al. New Eng. J. Med. 3251132-1136 (1991)). Nevertheless, the mechanism of progression fromchronic gastritis to malignant disease remains unclear, and therelationship of intestinal metaplasia, H. pylori infection and thedevelopment of cancer continues to be controversial. Moreover, while anassociation between gastric cancer and infection with H. pylori hasrecently been established, the cell of origin for gastric adenocarcinomais controversial. This does not establish a mechanism between thebacteria and the cancer, and provides little or no guidance forcorrelating treatment of, or risk associated with, H. pylori as itrelates to development of gastric cancer.

It is therefore an object of the present invention to provide screeningmethods for early gastric cancer.

It is a further object of the present invention to provide means forassessing the degree of early gastric cancer and for screening andfollowing patients at risk for gastric cancer.

It is a still further object of the present invention to provide meansfor serological testing for patients at risk of gastric cancer.

SUMMARY OF THE INVENTION

It has been determined that a specific metaplastic lineage that containsimmunoreactivity for a trefoil polypeptide, spasmolytic peptide, isassociated with and gives rise to the vast majority of humanadenocarcinomas. The identification of this Spasmolytic PolypeptideExpressing Metaplasia (SPEM) is a major factor for grading of biopsiesof the stomach to assess risk for gastric cancer. It also forms thebasis of a method for serological screening for those at risk forgastric cancer. In a preferred embodiment, antibodies to spasmolyticpeptide (hSP) are used in immunostaining of biopsies of gastric tissueobtained by endoscopy for grading biopsies. Those patients having thesecells, characterized by a morphology more typical of a type of cellpresent normally in the intestine and not stomach, Brunner's glandcells, are at risk of developing adenocacinoma. Since these cellsexpress hSP, antibodies or nucleic acid probes hybridizing to mRNAencoding hSP, can be used for rapid detection of the cells in tissuebiopsies. The antibodies can also be used in serological tests forscreening and following patients at risk for gastric cancer. Incombination with evidence of previous or present invention with H.pylori, the tests are predictive of the likelihood of developingadenocarcinoma.

DETAILED DESCRIPTION OF THE INVENTION

Metaplastic cell lineages arising in response to chronic injury areprecursors for the evolution of dysplasia and adenocarcinoma. Thissequence is well characterized in the case of the Barrett's epithelium,a columnar specialized intestinal metaplasia in the distal esophagus ofpatients with esophageal reflux (Haggitt, R. C. Hum. Pathol. 25, 982-993(1994)). While a subtype of intestinal metaplasia has been associatedwith gastric adenocarcinoma (Dixon, et al. Am. J. Surg. Pathol. 20,1161-1181 (1996); Filipe et al. Int. J. Cancer. 57, 324-329 (1994));Correa, P. Cancer Res. 48, 3554-3560 (1988)), the link between theselineages and the evolution of gastric adenocarcinoma has not been clear.It has now been determined that there is an association between SPEM,detectable in biopsies based on the presence of cells having amorphology similar to Brunner's gland cells, and adenocarcinoma.Although normal cells in the stomach, such as mucus neck cells, expresshSP, these cells are not predictive of adenocarcinoma.

I. Methods and Reagents for Screening

A. Histology of the Gastric Tissues

Presently, gastric mucosal biopsies are fixed in formalin and embeddedin paraffin. Microtome sections of tissues are then stained withhematoxylin and eosin. These stained slides are examined for loss ofparietal cells (oxyntic atrophy), ulceration, inflammatory infiltrates,and alterations in cell lineages including increased numbers of surfacemucous cells (foveolar hyperplasia), the presence of goblet cells(intestinal metaplasia), as well as dysplasia and adenocarcinoma.Dysplasia and adenocarcinoma are judged by changes in nuclearmorphology, loss of cytoplasmic space, loss of polarity and invasion ofsubmucosa or vasculature. Brunner's glands are not present in the normalstomach but can be observed in the duodenum.

B. Markers of SPEM

Small peptides displaying a cysteine-rich module (termed P-domain ortrefoil motif) form a group of peptides, including BCEI, expressed fromthe pS2 gene; hITF, expressed from the TFF3 gene; and hSP, expressedfrom the SML1 gene. These peptides are abundantly expressed at mucosalsurfaces of specific tissues and are associated with the maintenance ofsurface integrity. (Schmitt, et al., Cytogenet. Cell Genet. 72(4),299-302 (1996)). Human spasmolytic peptide (hSP) was identified byRomasetto, et al., EMBO J., 9(2), 407-414 (1990), based on homology topancreatic spasmolytic polypeptide, sequenced and determined to beseparately encoded on the genome from pS2. The gene sequence and aminoacid sequences of hSP can be obtained from GenBank, accession number1477545. Both are present in normal stomach epithelium. The patterns andtiming of the expression of the trefoil peptides are different from eachother. It is thought that S2 plays an important role in theproliferation of intestinal epithelial cells during the acute phase ofmucosal ulceration, whereas ITF may be involved in differentiation ofthe cells, particularly to form goblet cells, during the recovery phaseof acute colitis. (Itoh, et al., Biochem. J. 318(Pt 3), 939-944 (1996)).Immunostaining for SP in the intact mucosa has been determined to beconfined to the mucous neck cells, but following exposure to stress itwas significantly enhanced and occurred also in the cells of the basalregion of gastric glands, as reported by Konturek, et al., Regul. Pept.68(1), 71-79 (1997). Konturek, et al. (1997) proposed that SP plays animportant role in healing of stress-induced gastric lesions, possibly bythe acceleration of the mucosal repair, the enhancement of mucosal bloodflow and the inhibition of gastric secretion.

It has now been determined that SP is a marker of metaplastic cellshaving a morphology similar to those of Brunner's gland cells. Thesecells can be identified by histological examination. However, theidentification of SPEM with spasmolytic peptide immunostaining iseasier, more sensitive and rapid. Therefore, detection of metaplasticcells expressing SP provides a means for identification of those at riskwho would need further follow-up. Furthermore, since the SPEM lineage isoften extensive, quantitation of serum spasmolytic polypeptide levels byeither radioimmunoassay or ELISA should be useful to stratify patientsat risk and provide a serological method for identifying and followingpatients at risk for developing adenocarcinoma.

SPEM can be detected using antibodies or antibody fragments prepared bystandard techniques. The studies described in the examples wereperformed with a mouse monoclonal IgM anti-human spasmolytic polypeptidedeveloped by Drs. Richard Poulsom and Nicholas Wright at the ImperialCancer Research Fund, London, UK. Antibodies specifically directedtowards utility in RIA and ELISA have also been developed. Eithermonoclonal or polyclonal antibodies can be used. Antibodies can belabelled using any detectable marker, including radiolabels, fluorescentlabels, dyes, enzyme-chromogenic substrate systems, and other meanscommercially available.

SPEM can also be detected using nucleic acid probes which hybridizeunder standard hybridization conditions, as described for example, byManiatis, et al. Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory), to mRNA encoding SP. These can be labeled usingstandard labelling techniques for detection of bound nucleic acid.

Alternatively, or in addition, other markers of these cells can be usedto screen for the presence of SPEM in gastric tissue biopsies.

For serological assay of SP, serum would be obtained from fastingpatients. SP levels would be determined using either radioimmunoassay orenzyme-linked immunoassay. A standard curve would be used for knownamounts of recombinant SP (Lars Thimm, Novartis Corporation). Patientswith elevated levels of SP in serum would be investigated for thepresence of SPEM by endoscopy. Alternatively, patients with elevatedserum SP and documented SPEM could be followed following treatment of H.pylori by serial serum determinations of SP.

C. Detection of H. pylori Infection

Since H. pylori is known to be associated with an increased incidence ofadenocarcinoma of the stomach, efficacy of screening can be furtherenhanced by testing for previous or present H. pylori infection. H.pylori infection would be determined by either CLO test at the time ofbiopsy or H. pylori serology with the same sample used for SPdetermination.

II. Patients to be Screened and Screening Procedures

SP detection, such as immunohistochemistry, can be used to determine thepresence of SPEM in endoscopic biopsies as well as brushings obtainedeither through endoscopy or blind per oral intubation.

As noted above, gastric cancer screening, especially in Asian 25countries, is a major focus for clinical care. Presently, the onlyimpact of medicine on gastric cancer is through early detection of lowgrade tumors and early resection for cure. High grade tumors haveuniformly dismal prognosis with median survival less than one year. Nosignificant effect of adjuvant chemotherapy has been noted. The additionof spasmolytic polypeptide immunostaining of biopsies and theidentification of SPEM provides a means for identifying those at riskfor developing cancer in the future. Similarly, the use of spasmolyticpeptide serology should provide a blood test for identification of thoseat risk. Thus, the use of spasmolytic polypeptide immunostaining couldsignificantly decrease the number of screening endoscopies, focusscreening endoscopies, and, through serology testing, facilitatescreening of large populations a risk in Asia and other countries withhigh cancer incidence.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Background

Wang and colleagues examined the influences of chronic Helicobactergastritis using Helicobacter felis to infect the gastric epithelium ofC57BL/6 mice (Wang, et al. Gastroenterology. 114, 675-689 (1998)).Infection with H. felis produced a chronic gastritis with pathologicfeatures similar to human infection with H. pylori including marked lossof parietal and chief cell populations, in addition to elaboration of anaberrant mucous cell lineage (Lee, et al. Gastroenterology. 99,1315-1323 (1990); Fox, et al., Gastroenterology. 110, 155-166 (1996)).Wang, et al. (1998) reported that an aberrant metaplastic cell lineagewith morphological characteristics similar to Brunner's glands of theduodenum develops in the fundic mucosa of mice infected with H. felis.This metaplastic lineage expresses the trefoil peptide spasmolyticpolypeptide (SP). This expanded lineage stained with antibodies againstspasmolytic polypeptide (SP), a trefoil peptide secreted from mucousneck cells in the normal fundic mucosa (Wang, et al. (1998); Thim, L.FEBS Lett. 250, 85-90 (1989)). Importantly, the H. felis-inducedSP-expressing metaplastic (SPEM) lineage did not show morphologicalcharacteristics of mucous neck cells, but rather demonstrated morphologymore reminiscent of Brunner's glands of the duodenum (Wang (1998)).

Hypothesis

Given the results in mice, studies were designed to investigate whetherH. pylori infection would induce a similar aberrant SP-expressinglineage in human fundic mucosa. Given the epidemiological association ofHelicobacter sp. infection with gastric cancer, it was hypothesized thatthis SP-expressing metaplastic (SPEM) lineage may represent a precursorto or appear commensurate with gastric adenocarcinoma.

Summary of Results

The results of these studies showed that the SPEM lineage was present in65% of fundic biopsies from patients with fundic H. pylori-associatedgastritis, but was absent in biopsies of fundic mucosa from patientswithout H. pylori infection. In a review of archival samples from 22resected gastric adenocarcinomas, the SPEM lineage was found in 91% ofcases, typically located in mucosa adjacent to the carcinoma or areas ofdysplasia. Importantly, 56% of samples showed SP immunoreactivity withindysplastic cells. These data indicate not only a strong associationbetween the SPEM lineage and gastric adenocarcinoma, but that SPEM cellsrepresent the metaplastic lineage responsible for development of thistumor in patients with H. pylori.

EXAMPLE 1 Detection of SP Levels in Gastric Fundic Biopsies

Ten fundic biopsies from H. pylori negative patients and 17 biopsiesfrom patients with H. pylori colonization in the fundus were examined.All biopsies demonstrated H/K-ATPase-immunostaining parietal cells andcontained no gastrin-immunoreactive cells, confirming the authenticityof fundic mucosal biopsies. In all of the H. pylori-infected biopsiesspecimens, the presence of H. pylori was confirmed by Giemsa staining oftissue sections. The histological characteristics of these biopsies aresummarized in Table I.

TABLE I Histological analysis of fundic biopsies from H. pylori (Hp)negative and H. pylori positive patients. HP Negative HP Positive AbsentPresent Absent Present Foveolar hyperplasia 10 0 4 13 Oxyntic atrophy 100 6 11 Mononuclear infiltrates 8 2 1  16* SPEM 10 0 6 11

*3 of 16 H. pylori (Hp) positive patients demonstrated organizedlymphoid aggregates. SPEM was assessed in sections stained for hSP.

Foveolar hyperplasia was assessed in sections stained for pS2 as thepresence of surface cells in greater than 25% of the gland length.Oxyntic atrophy was assessed in sections stained for H/K-ATPase as adecrease in parietal cell density to less than half the gland length.Mononuclear infiltrates were assessed in H&E stains.

Gastric fundic biopsies were stained for SP. 10 μm sections fromparaffin-embedded biopsies of fundic mucosa from H. pylori uninfectedand H. pylori infected patients were stained for hSP with a monoclonalmurine anti-HSP Elia, et al. Histochem. J. 26, 644-647 (1994)). Dewaxedparaffin sections were blocked with 5% goat serum in phosphate bufferedsaline and then incubated with anti-hSP (1:50) for one hour at roomtemperature. Indirect immunohistochemical detection was then performedthrough incubation with biotinylated anti-mouse IgG,streptavidin-conjugated alkaline phosphatase and finally Vector Redchromogen substrate (Vector Laboratories, Burlingame, Calif.). Forconfirmation of the veracity of biopsies as fundic mucosa, serialsections were also stained with monoclonal antibodies against theH/K-ATPase (1:5000, the gift of Dr. Adam Smolka, Medical University ofSouth Carolina, a marker of parietal cells) and gastrin (undiluted,Zymed, a marker of antral G-cells).

The presence of H. pylori was confirmed in all biopsies with Giemsastaining. All biopsies demonstrated immunoreactive parietal cellswithout the presence of G-cells). In non-infected patients, staining wasconfined to normal appearing mucous neck cells. In H pylori-infectedpatients, nodular aggregates of hSP-staining cells with thecharacteristics of Brunner's gland cells (SPEM) were observed in 65% ofbiopsies. No SPEM was observed in fundic biopsies from non-infectedpatients.

In non-infected patients, fundic biopsies demonstrated only normalappearing SP-immunoreactive mucous neck cells. In contrast, 65% ofbiopsies from H. pylori infected biopsies exhibited aberranthSP-immunoreactive SPEM cells. The lineage showed a morphology morecharacteristic of Brunner's gland cells. Most cells were present asnodular formations in the deeper portions of the metaplastic glands.Foveolar hyperplasia was present in 70% of H. pylori infected biopsies(Table I). Significant mononuclear infiltrate was present in 95% ofbiopsies with 18% of the biopsies demonstrating organized lymphoidaggregates. These results indicated that the SPEM lineage was present inthe fundic mucosa of many patients with H. pylori-associated fundicgastritis.

EXAMPLE 2 Association of SPEM with Gastric Adenocarcinomas

Since chronic H. pylori infection is associated with the development ofadenocarcinoma, archived tissues from resected gastric adenocarcinomasin patients were examined. All of the resections were for tumors of thefundus or fundal/antral border.

Ten μm sections of paraffin-embedded mucosa from regions adjacent togross adenocarcinoma were examined for immunostaining with hSPantibodies as described above. Prominent regions of SPEM were observedin mucosa underlying regions of foveolar hyperplasia. The lineage wasalways observed near the bases of glands. As noted in the biopsies, theSPEM cells were large with extensive numbers of immunoreactive granules.In an adjacent region, hSP staining of normal-appearing mucous neckcells was observed. Parietal cells were also observed in these sections.Adjacent sections were stained with anti-PSTI (1:200) with staining asdescribed above except that sections underwent retrieval using citratebuffer in a microwave and horseradish peroxidase-conjugated secondaryantibodies were used for detection with diaminobenzidine chromogen.While distinct PSTI immunoreactive mucous neck cells were observedwithin the mucosa, the SPEM lineage cells at the right side of thesection showed no PSTI immunoreactivity. Adjacent sections were alsostained with anti-PS2 (1:50) as described above. While pS2immunostaining was observed in regions of surface cell foveolarhyperplasia, SPEM cells did not stain for pS2. Toluidine blue stainingof a 0.5 μm section of glutaraldehyde fixed tissue from a resectionspecimen demonstrates the numerous granules in SPEM cells. Note thepresence of residual parietal cells in some SPEM-containing glands,confirming the presence of the lineage in fundic mucosa. Electronmicroscopic examination demonstrates the presence of multiple mucousgranules within the SPEM cells along with the characteristic expandedapical membrane surface without microvilli.

In summary, twenty patients (91%) demonstrated the SPEM lineage withintheir resection specimens. No evidence of the lineage was observed intwo patients with diffuse infiltrating adenocarcinoma. In mostspecimens, a prominent homogeneous cell population expressing hSP wasnoted dominating greater than half the lower length of the fundicglands. These SPEM cells possessed an abundance of cytoplasmic mucingranules and a wide apical surface. Branching of SPEM-containing glandswas observed in many specimens. Most glands displayed varying degrees ofoxyntic cell atrophy, however there was more variability in expansion ofthe surface cell compartment, with 68% of patients having foveolarhyperplasia. Normal mucous neck cell staining with hSP and parietalcells were observed in regions adjacent to SPEM-containing regions.Goblet cell-containing intestinal metaplasia was only observed in 10% ofspecimens.

Resection specimens were stained with several lineage-specific markersto characterize the origin of the SPEM lineage more completely.Increased expression of TGFα has been noted previously in foveolar cellsof patients with Menetrier's disease and hypertrophic lymphocyticgastritis (Dempsey, et al. Gastroenterology. 103, 1950-1963 (1992);Bluth, et al. Hum. Pathol. 26, 1333-1340 (1995)). However, clearoverexpression of TGFα in the foveolar regions of the mucosa adjacent totumor was not observed. In addition, the SPEM lineage did not stain withantibodies against TGFα. In all cases, there was no evidence ofenterochromaffin-like (ECL) cell hyperplasia, although scatteredchromogranin A immunoreactive cells were observed. The SPEM lineage didnot stain with antibodies against chromogranin A. While antibodiesagainst pancreatic secretory trypsin inhibitor (PSTI) did stainscattered normal appearing mucous neck cells (McKenzie, et al. Am. J.Physiol. 273, G112-G117 (1997)), no staining of the lineage wasobserved. While antibodies against the trefoil peptide pS2 did stainsurface mucous cells in areas of foveolar hyperplasia, no staining wasobserved in the SPEM lineage. Previous investigations have suggestedthat Intestinal Trefoil Factor (ITF), which is absent from the normalgastric mucosa, is unregulated following injury (Podolsky, et al. J.Biol. Chem. 268, 6694-6702 (1993); Alison, et al. J. Pathol. 175,405-414 (1995)). While occasional cells expressing ITF mRNA could beidentified in regions of intestinal metaplasia, no ITF expression couldbe documented in the SPEM lineage. Finally, examination of SPEM in boththick and thin plastic sections demonstrated the presence of abundantmucous granules and a broadened apical surface in these cells. Theseultrastructural characteristics are similar to those of SPEM cellsobserved in H. felis-infected C57BL/6 mice (Wang (1998)). All of thesefindings indicate that the SPEM lineage is not directly derived from anormal gastric lineage. Rather it appears to represent a metaplasticlineage and is differentiable from both normal mucous neck cells and thegoblet cell-containing type metaplasia previously associated withchronic H. pylori infection and atrophic gastritis (Dixon, et al.(1996)).

As noted above, the SPEM lineage was observed in apposition with bothdysplastic lineages as well as adenocarcinoma. hSP staining was observedin both SPEM as well as in contiguous regions of dysplasia in 49% ofresection samples. Immunoreactivity was detectable in a region ofadenocarcinoma adjacent to the hSP-irnmunoreactive cells. At highermagnification, dysplastic epithelial cells appear to be contiguous withSPEM cell-containing glands. Importantly, however, in 59% of theresections, hSP immunoreactivity was present in cells within regions ofsevere dysplasia or carcinoma in situ. In several cases regions ofSP-expressing dysplastic cells were noted extending from regions of SPEMlineage. While regions containing the SPEM lineage did not showsignificant labelling with Ki-67 and PCNA antibodies, the dysplasticareas showed prominent nuclear labelling in addition to characteristicchanges in cell morphology. These results indicate that the SPEM lineagemay represent a metaplastic precursor for the development of dysplasiaand adenocarcinoma.

Previous investigations have focused on the association of Type IIIintestinal metaplasia with the development of adenocarcinoma. Despitethis association, it is less clear that neoplastic cells actually arisefrom areas in goblet-cell containing intestinal metaplasia. Of note,while intestinal metaplasia and foveolar hyperplasia are oftenconspicuous in atrophic gastritis in association with H. pyloriinfection, gastric adenocarcinomas usually develop deep in the glands asnodular lesions that underlie the regions of intestinal metaplasia orfoveolar hyperplasia. The identification of the SPEM lineage supportsthe proposal that this metaplastic candidate precursor is located inproximity with the putative site of neoplastic transition.

It is not clear whether hSP itself might contribute to the developmentof adenocarcinoma. hSP has generally been associated with cytoprotectionin the gastric mucosa (McKenzie, et al., (1997); Tanaka, et al. Am. J.Physiol. 272, G1473-G1780 (1997); Babyatsky, et al. Gastroenterology.110, 489-497 (1996)). Homologous deletion of the gene for the gastricsurface cell trefoil protein pS2 leads to the development ofintramucosal carcinomas (Lefebvre, et al. Science 274, 259-262 (1996)),however the lineage responsible for these lesions is unclear. It is morelikely that the expression of SP is a functional marker of thismetaplastic lineage with Brunner's gland morphology. A metaplasia withBrunner's gland morphology expressing hSP has been associated with acuteand chronic injury of the gastrointestinal mucosae (Wright, et al.Nature. 343, 82-85 (1990); Ahnen, et al. J. Pathol. 173, 317-326(1994)). Nevertheless, the ulcer-associated cell lineage (UACL) appearsto constitute a separate entity, since SPEM does not express ITF.Nevertheless, one can not rule out that SPEM might evolve from UACL inthe gastric mucosa.

The presence of a well differentiated metaplastic lesion as a precursorof adenocarcinoma is not without precedence. Esophageal adenocarcinomadevelops from dysplastic transition within the metaplastic columnarBarrett's epithelium in the distal esophagus (Reid, et al.Gastronenterology. 102, 1212-1219 (1992); Miros, et al. Gut. 32,1441-1446 (1991)). As with the SPEM lineage in the stomach, theBarrett's epithelium shows low proliferative indices. Dysplasia andadenocarcinoma only develop in a minority (10-15%) of patients. Thesestudies indicate that, while the SPEM lineage is commonly associatedwith fundic H. pylori gastritis, only a minority of patients willprogress to dysplasia and adenocarcinoma. It is therefore likely thatfurther events must occur to elicit the development of the neoplastictransition. Thus, as recognition and monitoring of Barrett's epitheliumis the mainstay of surveillance for the development of esophagealadenocarcinoma (Robertson, et al, M. Br. J. Surg. 75, 760-763 (1988)),so surveillance of patients at risk for gastric adenocarcinoma shouldfocus attention on this metaplastic precursor lineage expressing SP.

In summary, these data support the hypothesis that the SPEM lineage is alink between chronic H. pylori gastritis and dysplasia leading togastric adenocarcinoma.

We claim:
 1. A method for screening for Spasmolytic PolypeptideExpressing Metaplasia (SPEM) in metaplastic cells having Brunner's glandmorphology with abundant mucous granules and a broadened apical surface,wherein the SPEM is found in apposition with gastric adenocarcinomacells in an adenocarcinoma patient, the method comprising detecting thepresence of metaplastic cells expressing spasmolytic peptide which haveBrunner's gland morphology in gastric tissues isolated from the patient.2. The method of claim 1, wherein the cells expressing spasmolyticpeptide are detected using reagents specific for spasmolytic peptide. 3.The method of claim 2, wherein the spasmolytic peptide is detected usingantibody to spasmolytic peptide.
 4. The method of claim 1, wherein thespasmolytic peptide is detected in a biopsy of gastric tissues.
 5. Themethod of claim 1, wherein the screening is performed prior toconfirmation of development of adenocarcinoma.
 6. A method for screeningfor gastric adenocarcinoma comprising detecting the presence ofmetaplastic cells expressing spasmolytic peptide which have Brunner'sgland morphology with abundant mucous granules and a broadened apicalsurface in gastric tissues isolated from the patient.
 7. The method ofclaim 6, wherein the cells expressing spasmolytic peptide are detectedusing reagents specific for spasmolytic peptide.
 8. The method of claim7, wherein the spasmolytic peptide is detected using antibody tospasmolytic peptide.
 9. The method of claim 6, wherein the spasmolyticpeptide is detected in a biopsy of gastric tissues.
 10. The method ofclaim 6, wherein the screening is performed prior to confirmation ofdevelopment of adenocarcinoma.